Regulation of Outside-in Integrin Signaling in Platelets

血小板中由外而内整合素信号传导的调节

• 批准号: 8213150
• 负责人: SANFORD J SHATTIL
• 金额: $ 38.63万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8213150
• 关键词: Actinin; Actins; Address; Adhesions; Adhesives; Affect; Agonist; Binding; Biological; Biological Assay; Biological Models; Bioluminescence; Bleeding time procedure; Blood Cells; Blood Platelets; Blood Vessels; C-terminal; Calpain; Carotid Arteries; Cell model; Cells; Co-Immunoprecipitations; Collagen; Complex; Cytoplasmic Tail; Cytoskeletal Modeling; Cytoskeletal Proteins; Cytoskeleton; Defect; Dissociation; Drosophila genus; Drug Delivery Systems; Energy Transfer; Event; Exhibits; Extracellular Matrix; Fibrinogen; Fibrinogen Receptors; Fibroblasts; Fluorescence; Fluorescence Resonance Energy Transfer; Gene Targeting; Goals; Hematological Disease; Hemostatic function; Human; Image; Immunoprecipitation; Injury; Integrins; Investigation; Knock-out; Laboratories; Laser injury; Lead; Life; Ligands; Mediating; Megakaryocytes; Mesentery; Modeling; Molecular; Monitor; Mus; PTPN1 gene; Phase; Phosphoric Monoester Hydrolases; Phosphotransferases; Platelet aggregation; Principal Investigator; Process; Protein Tyrosine Phosphatase; Proteins; Radiation Chimera; Recombinants; Recruitment Activity; Regulation; Reporter; Role; SH3 Domains; SRC gene; Signal Transduction; Site; Specificity; System; Tail; Techniques; Testing; Thrombosis; Thrombus; Tissues; Tyrosine Phosphorylation; Vinculin; Work; adapter protein; arteriole; base; in vivo; insight; mutant; novel; polymerization; polypeptide; programs; protein protein interaction; receptor; reconstitution; research study; response; src-Family Kinases; von Willebrand Factor

Following vascular injury, adhesive ligands such as fibrinogen and von Willebrand factor engage integrin allb/?3 to effect platelet aggregation and spreading during hemostasis and thrombosis. These responses are triggered by ligand-mediated allb/?3 clustering, which initiates "outside-in" signals to reorganize the actin cytoskeleton. Recent work from this project has established that outside-in signaling in platelets requires Src family tyrosine kinases (SFKs), c-Src in particular, which bind to /?3 and are activated by allb/?3 clustering in a manner dependent on PTP-1B, a protein tyrosine phosphatase. Here, three major unresolved questions will be asked concerning the molecular basis of outside-in signaling in platelets and its biological consequences. First, do direct interactions between integrins and SFKs represent a general mechanism for spatio-temporal initiation of outside-in signaling in platelets? Since platelets contain five different integrins and at least six different SFKs, this possibility will be evaluated by co-immunoprecipitation techniques, by bimolecular fluorescence complementation imaging in live cells, and by localization of Src activation in live cells using a FRET-based reporter. In addition, integrin/SFK interactions will be assessed in Drosophila cells to determine the extent to which direct activation of SFKs by integrins is an evolutionarily conserved process. Second, how does PTP-1B activate c-Src downstream of integrins? The mechanism by which PTP-1B is recruited to the allb/?3/c-Src complex, and possibly to other integrin/SFK complexes, will be evaluated in platelets and model cell systems, focusing on the possible role of adapter proteins. In addition, the effect of integrin clustering on PTP-1B catalytic activity will be determined. Third, does selective disruption of outsidein signaling affect thrombus formation in vivo? Here arterial thrombosis will be studied in novel gene-targeted mice predicted to have selective defects in the interaction of c/llb/83 with c-Src or other SFKs, or defects in downstream events required for actin reorganization. Altogether, these studies will provide molecular insights into how outside-in integrin signaling is initiated and establish the extent to which this process regulates platelet function in vivo. Thus, this line of investigation may lead to identification of new anti-thrombotic drug targets and serve as a paradigm for integrin signaling in other blood cells.

血管损伤后，粘附配体如纤维蛋白原和血管性血友病因子与整合素结合， allb/？3在止血和血栓形成过程中影响血小板聚集和扩散。这些响应是 由配体介导的allb/？3成簇，启动“由外向内”信号以重组肌动蛋白 细胞骨架该项目最近的工作已经确定，血小板中由外向内的信号传导需要Src 家族酪氨酸激酶（SFKs），特别是c-Src，其结合到/？3和激活的allb/？3.集群 一种依赖于PTP-1B的方式，PTP-1B是一种蛋白酪氨酸磷酸酶。在这里，三个主要的悬而未决的问题 将被问及关于血小板中由外向内信号传导的分子基础及其生物学特性， 后果首先，整合素和SFK之间的直接相互作用是否代表了整合素与SFK之间的一般机制？ 血小板中由外向内信号的时空启动？由于血小板含有五种不同的整合素 和至少六种不同的SFK，这种可能性将通过免疫共沉淀技术进行评估， 活细胞中的双分子荧光互补成像，以及通过活细胞中Src激活的定位， 细胞使用FRET为基础的报告。此外，将在果蝇细胞中评估整合素/SFK相互作用 以确定整联蛋白直接激活SFKs在何种程度上是进化上保守的过程。 第二，PTP-1B如何激活整合素下游的c-Src？PTP-1B的作用机制 被招募到allb/？3/c-Src复合物，以及可能与其他整联蛋白/SFK复合物的关系，将在 血小板和模型细胞系统，侧重于衔接蛋白的可能作用。此外， 将测定整联蛋白簇集对PTP-1B催化活性的影响。第三，选择性地破坏外部环境 信号传导影响体内血栓形成？在这里，动脉血栓形成将在新的基因靶向研究， 预测在c/IIb/83与c-Src或其它SFK的相互作用中具有选择性缺陷的小鼠，或在c/IIb/83与c-Src或其它SFK的相互作用中具有选择性缺陷的小鼠， 肌动蛋白重组所需的下游事件。总之，这些研究将提供分子见解 整合素信号是如何启动的，并确定该过程调节的程度。 体内血小板功能。因此，这一研究路线可能会导致新的抗血栓药物的发现 靶向并作为其他血细胞中整合素信号传导的范例。